1. Field of the Invention
The present invention relates to benzoxazole-based marker dyes for UV and short wave excitation having a large Stokes shift.
2. Discussion of Background Information
Fluorescence-based markers have been used for decades in biological, biotechnological and medical research and also in medical diagnostics [Brinkley M., A Brief Survey of Methods for Preparing Protein Conjugates with Dyes, Haptens and Cross-Linking Reagents, Bioconjugate Chem, 3 (1992) 2-12; Waggoner A., Covalent Labeling of Proteins and Nucleic Acids with Fluorophores, Meth. Enzymol., 246 (1995) 362-373]. Originally, chromophores known from the field of textile and sensitizer dyes were derivatized to optimize them for applications in the aqueous physiological milieu. Developments here were focused in particular on solubility in water and also a high quantum yield in aqueous solution, the starting point for such development work usually being a film dye [Poppe E.-J., Sensibilisierungsfarbstoffe mit sauren N-Substituenten, Z. wiss. Photographie, 63 (1969) 149-158 and references cited therein].
Classes of dyes frequently used here are the xanthylium (fluorescein, rhodoles, rhodamines), cyanine (carbo, di-carbo and tri-carbo), dipyrrhomethene (BoDiPy) and coumarin chromophores, although the diversity of dyes is not restricted to the types referred to [Panchuk-Voloshina N. et al., Alexa Dyes, a Series of New Fluorescent Dyes that Yield Exceptionally Bright, Photostable Conjugates, J. Histochem. Cytochem., 47 (1999) 1179-1188].
Proceeding from commercially available light sources such as mercury and xenon burners and also tungsten lamps and constrained by the spectral limitation to the light which is visible to the human eye, fluorescent markers were first developed for chromophores emitting between 400 nm and about 650 nm.
The advent of lasers as excitation light sources opened up completely new possibilities of spectral combination far beyond the visible region on the dye side as well as for the optical detection to be realized on the equipment side, owing to the high intensity of lasers leading to a significantly enhanced sensitivity on the part of the measuring instruments. In addition to conventional lasers, the virtually monochromatic light-emitting diodes (LEDs) and solid-state laser diodes [Imasaka T., Diode lasers in analytical chemistry, Talanta, 48 (1999) 305-320] are still becoming more and more important in the fluorescence-based detection for bioanalytical applications.
The motivation to glean more and more information from bioanalytical procedures finds expression in multicolor analyses that have become part of routine laboratory practice, for example in flow cytometry [Lee L. G., NEAR-IR Dyes in Three Color Volumetric Capillary Cytometry: Cell Analysis With 633- and 785-nm Laser Excitation, Cytometry, 21 (1995) 120-128], DNA sequencing and various PCR methods (Roche's LightCycler). Dye combinations were initially used here to deliver spectrally distinguishable signals on excitation by one and the same (monochromatic) light source via energy transfer from a donor to various acceptors. Examples thereof are the DNA sequencers from Amersham (now GE Healthcare) and ABI (now Life Technologies), which came on the market at the end of the 1990s.
DNA and RNA in the present description refer to deoxyribonucleic acid and ribonucleic acid respectively.
An alternative approach to multiplex applications is to use dyes that permit spectral differentiation without energy transfer between donor and acceptor. Examples thereof are found in the MegaStokes dyes known since about 2002 (EP 1 318 177 B1, EP 1 535 969 B1). These are preferentially tailored to an excitation wavelength between 470 nm (cyan LEDs) and 500 nm (488 nm Ar-ion lasers) to best meet the then state of the art in excitation light sources.
Similarly the combination of two or more excitation light sources with more than one chromphore per light source is now established prior art (Solexa, WO2007/135,368A2). A typical application here is Next Generation Sequencing (NGS).
The availability of light-intensive, short-wave excitation sources such as UV-LEDs or violet laser diodes also widened the base of suitable fluorophores for bioanalytical applications. One example thereof is Pacific Orange from Molecular Probes (U.S. Pat. No. 8,158,801), which similarly to the MegaStokes™ dyes permitted multicolor analyses in combination with coumarin-based chromophores, albeit on excitation at around 400 nm.
The invention has as its object the provision of benzoxazole-based fluorescent markers having a large Stokes shift, a high photo and storage stability and also a high fluorescence quantum yield.